Immunodiagnostic procedures for human fascioliasis use antigenic mixtures, obtained from live parasites. Therefore, the specificity and sensitivity may be compromised because of the presence of irrelevant contaminants of parasite or host origin. Use of a purified antigen in the immunodiagnostic protocols will be important in aiding the diagnosis of human fascioliasis. We propose to establish the serologic relevance of antigens present in metabolic products and in somatic extracts of Fasciola hepatica. Currently, we have identified a 44 kDa polypeptide in adult worm homogenates that is recognized by sera from experimental infections in cattle. The 44 kDa polypeptide is also recognized by antibodies in sera obtained from confirmed human cases. This polypeptide is not recognized by sera collected from non-infected cattle and rabbits, healthy individuals and from sera collected from humans infected with Schistosoma mansoni, S. haematobium, Leishmania donovani, Entoameba histolytica, G. lamblia, T. gondii, H. nana and Strongyloides. Purification of identified target antigens will be conducted by conventional gel filtration, anion-exchange chromatography and preparative electrofocusing. The target antigens will be further fractionated using ion-exchange high performance liquid chromatography (HPLC). Peptide mapping of the antigens will be accomplished by treatment of polypeptides with enzymes, including trypsin beta-amylase and proteinase K. The digests will be isolated by HPLC and evaluated for reactivity in the biotin-avidin enzyme-linked immunosorbent assay (ELISA) and western blotting. Biochemical studies will include determination of molecular size, molecular weight and isoelectric point. These characterizations will be complemented with the isolation of relevant genes. Thus, the identification of immunodominant epitopes will provide valuable clinical information in the diagnosis of F. hepatica in humans. In addition, one graduate student will be properly trained in immunochemistry and stimulated to establish an independent research in immunochemistry program in Puerto Rico.